title: Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae.
abstract: Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.
authors.affiliation: ANSES Laboratoire de la Santé des Végétaux, Unité de Mycologie, IFR110 EFABA, Domaine de Pixérécourt, BP 90059, 54220 Malzéville, France. anne-laure.boutigny@anses.fr
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Boutigny
authors.forename: Cécile
authors.initials: C
authors.lastname: Guinet
authors.forename: Agathe
authors.initials: A
authors.lastname: Vialle
authors.forename: Richard C
authors.initials: RC
authors.lastname: Hamelin
authors.forename: Axelle
authors.initials: A
authors.lastname: Andrieux
authors.forename: Pascal
authors.initials: P
authors.lastname: Frey
authors.forename: Claude
authors.initials: C
authors.lastname: Husson
authors.forename: Renaud
authors.initials: R
authors.lastname: Ioos
mesh_terms: D001487:Basidiomycota / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005060:Europe; D009172:Mycology / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D032107:Populus / Q000382:microbiology; D011790:Quarantine; D012331:RNA, Fungal / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012339:RNA, Ribosomal, 28S